This invention relates to nucleic acid and amino acid sequences of a transducin beta-1 subunit and to the use of these sequences in the diagnosis, prevention, and treatment of diseases associated with cell proliferation.
Membrane-bound GTP-binding (G) proteins mediate signal transduction in a variety of cell systems. Although, the exact mechanisms of G protein action are still under investigation, G proteins appear to involve effectors located in the plasma membrane as well as other parts of the cell. WD-40 proteins contain a loosely conserved repeat of approximately 40 amino acids separated by a Trp-Asp dipeptide sequence which may recur several times within each polypeptide. The conserved core of the WD-40 sequence, which usually ends with the amino acids Trp-Asp (WD), was first identified in the xcex2-subunit of the heterotrimeric G protein, transducin. Several dozen WD-40 proteins have since been identified, none are enzymes, and all seem to have regulatory functions (Neer, E. J. et al. (1994) Nature 371:297-300).
Many of the WD-40 proteins are homologs of xcex2-transducin and function in signal transduction pathways within the cytoplasm. WD-40 proteins may participate in complex formation, and may interact with other G protein subunits through the WD-40 region. The xcex2 subunit of G proteins enhances binding of the Gxcex1 subunit to receptors and assists in the assembly of the G-protein:receptor complex. The Gxcex2xcex3 subunit binds to and brings the xcex2-adrenergic receptor kinase, xcex2-ARK, to the receptor (Touhara, K. et al. (1994) J. Biol. Chem. 269: 10217-10220).
A number of WD repeat proteins are localized to the nucleus and function in the repression of transcription. These include Tup1, Hir1, and Met30 in S. cerevisiae; SCON2 in Neurospora crassa; extra sex combs and Groucho in Drosophila; COP1 in Arabidopsis thaliana; and Rec14 in Schizosaccharomyces pombe. These WD-40 proteins turn off a wide variety of genes, including those involved in segmentation, sex determination, and neurogenesis, and those involved in photomorphogenesis.
The WD repeat protein, Rec14, is essential for meiotic recombination in S. pombe. Mutations in the Rec14 gene reduce meiotic recombination by as much as a factor of 1000 on chromosomes I and III. The Rec14 gene contains two exons separated by a 53-bp intron. The spliced transcript encodes a protein of 302 amino acids and contains six WD repeat motifs common in the G-beta transduction family and in S. cerevisiae Rec103 protein. Rec103 mutations have no detectable effect on mitotic recombination. Based upon phenotypes and amino acid sequence similarities, it is possible that R is a functional homolog of S. cervisiae Rec103 (Evens, D. H. et al. (1997) Genetics 146:1253-1264). Autoantibodies occurring spontaneously in certain cancer patients are reagents for identifying cellular proteins, and it is the WD-40 motif in the beta-transducin proteins which target these antibodies.
The discovery of a new transducin beta-1 subunit and the polynucleotides encoding it satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention and treatment of diseases associated with cell proliferation, in particular, cancers and immune response.
The invention features a substantially purified polypeptide, transducin beta-1 subunit (TBS), having the amino acid sequence shown in SEQ ID NO:1, or fragments thereof.
The invention further provides an isolated and substantially purified polynucleotide sequence encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or fragments thereof and a composition comprising said polynucleotide sequence. The invention also provides a polynucleotide sequence which hybridizes under stringent conditions to the polynucleotide sequence encoding the amino acid sequence SEQ ID NO:1, or fragments of said polynucleotide sequence. The invention further provides a polynucleotide sequence comprising the complement of the polynucleotide sequence encoding the amino acid sequence of SEQ ID NO:1, or fragments or variants of said polynucleotide sequence.
The invention also provides an isolated and purified sequence comprising SEQ ID NO.2 or variants thereof. In addition, the invention provides a polynucleotide sequence which hybridizes under stringent conditions to the polynucleotide sequence of SEQ ID NO:2. The invention also provides a polynucleotide sequence comprising the complement of SEQ ID NO:2, or fragments or variants thereof.
The present invention further provides an expression vector containing at least a fragment of any of the claimed polynucleotide sequences. In yet another aspect, the expression vector containing the polynucleotide sequence is contained within a host cell.
The invention also provides a method for producing a polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment thereof, the method comprising the steps of: a) culturing the host cell containing an expression vector containing at least a fragment of the polynucleotide sequence encoding TBS under conditions suitable for the expression of the polypeptide; and b) recovering the polypeptide from the host cell culture.
The invention also provides a pharmaceutical composition comprising a substantially purified TBS having the amino acid sequence of SEQ ID NO:1 in conjunction with a suitable pharmaceutical carrier.
The invention also provides a purified antagonist of the polypeptide of SEQ ID NO:1. In one aspect the invention provides a purified antibody which binds to a polypeptide comprising the amino acid sequence of SEQ ID NO:1.
Still further, the invention provides a purified agonist of the polypeptide of SEQ ID NO:1.
The invention also provides a method for treating or preventing an immune response disorder comprising administering to a subject in need of such treatment an effective amount of an antagonist to TBS.
The invention also provides a method for treating or preventing a cancer comprising administering to a subject in need of such treatment an effective amount of an antagonist to TBS.
The invention also provides a method for detecting a polynucleotide which encodes TBS in a biological sample comprising the steps of: a) hybridizing the complement of the polynucleotide sequence which encodes SEQ ID NO:1 to nucleic acid material of a biological sample, thereby forming a hybridization complex; and b) detecting the hybridization complex, wherein the presence of the complex correlates with the presence of a polynucleotide encoding TBS in the biological sample. In one aspect the nucleic acid material of the biological sample is amplified by the polymerase chain reaction prior to hybridization.